The transforming growth factor-β (TGF-β) superfamily consists of an increasing number of molecules that regulate a variety of cellular processes such as growth, differentiation and oncogenesis. Members of the TGF-β superfamily have been classified into major family groupings which include TGF-β, bone morphogenic protein (BMP), growth and differentiation factor (GDF), inhibin/activin, mullerian inhibitory substance (MIS), glial derived neurotrophic factor (GDNF) and, more recently, macrophage inhibitory cytokine-1 (Bootcov et al., 1997). The involvement of the TGF-β superfamily in human pregnancy is indicated by the detection of TGF-β1, TGF-β2, TGF-β3,activin and inhibin in amniotic fluid and the localisation of TGF-β1, activin and inhibin to the placental villi (Graham et al., 1992; Petraglia et al., 1993a; Petraglia et al., 1992; Minami et al., 1992; Lang and Searle, 1994; Qu and Thomas, 1992; Altman et al., 1990; Canniggia et al., 1999; Wallace et al., 1997).
The TGF-β superfamily has been studied intensively because of their biological importance and therapeutic potential. Their biology and functions are well known and have been extensively reviewed (e.g. Miyazono et al., 1993; Wahl, 1992; and Roberts et al., 1993). They are potent chemotactic factors for macrophages and fibroblasts and generally inhibit cell proliferation, perhaps because of their role in differentiation. In the context of inflammation, TGF-β is a potent stimulator of fibroblasts, collagen and matrix protein synthesis, promotes angiogenesis, modulates expression of adhesion molecules and inhibits lymphocyte proliferation, production of some lymphokines and NK cell function. TGF-β proteins have also been heavily implicated in the pathogenesis of chronic inflammatory processes And mechanisms.
The TGF-β superfamily is also thought to perform multiple roles during pregnancy. The ability of the TGF-β isoforms to modulate cell-cell adhesion, cell migration and tissue remodelling has led some authors to suggest that these molecules may control trophoblast invasion and implantation in early pregnancy. Other possible roles include regulation of foetal growth and suppression of the maternal immune system. Placental cells are a major source of TGF-β superfamily molecules and are regulated by at least TGF-β1, TGF-β3, activin and inhibin. For example, activin suppresses the production of inhibin and enhances progesterone, human chorionic gonadotropin (hCG), and gonadotropin-releasing hormone (GnRE) by placental cells (Petraglia et al., 1989). Inhibin suppresses placental hCG, GnRH and activin-induced progesterone release (Petraglia et al., 1989), while TGF-β1 suppresses placental derived human placental lactogen (hPL) production. Activin and TGF-β3 have also been shown to have opposing effects in regulating extravillous trophoblast invasion in early pregnancy (Caniggia et al., 1997; Caniggia et al., 1999). These findings suggest that TGF-β1, TGF-β3, activin and inhibin regulate the growth and differentiation of the placenta in an autocrine manner. TGF-β1, activin and inhibin are also present in the embryo proper where they have been demonstrated to regulate growth and differentiation. In particular, TGF-β superfamily members are well known for their ability to promote mesoderm induction.
It has also been suggested that TGF-β superfamily proteins promote foetal survival. Experimental evidence suggests that the amniotic fluid concentration of the pro-inflammatory cytokines interleukin-1 (IL-1), IL-6, and tumour necrosis factor-α (TNF-α) rise during labour. Furthermore, pro-inflammatory cytokine production accompanying intrauterine infection has been associated with foetal rejection or preterm labour (Romero et al., 1992; Hillier et al., 1993; Opsjon et al., 1993). TGF-β1 and inhibin have been shown to suppress the production of pro-inflammatory cytokines from macrophages and lymphocytes respectively (Bogdan and Nathan, 1993; Petraglia et al., 1991) while activin has pro-inflammatory effects on macrophages and amnion (Nusing and Barsig, 1997; Petraglia et al., 1993b). This has led to the suggestion that TGF-β1 and inhibin promote foetal survival by suppressing the production of pro-inflammatory cytokines by the maternal immune system.
The present applicants have recently cloned and characterised a divergent member of the TGF-β superfamily, macrophage inhibitory cytokine-1 (MIC-1) (Bootcov et al., 1997), whose expression is associated with macrophage activation. In order to determine the nature of any role MIC-1 may play in pregnancy, the present applicants have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for MIC-1 quantification and used this to investigate the temporal relationship between human maternal serum MIC-1 concentrations and gestation age, and further, measured its concentration in amniotic fluid and placental extracts. In addition, the present applicants have conducted experimentation to delineate the origins of MIC-1 by assessing the capacity of a placental trophoblastic cell line (BeWo) to synthesise the cytokine. The results presented hereinafter shows that MIC-1 is able to promote foetal survival by suppressing the production of maternally-derived pro-inflammatory cytokines within the uterus. Consequently, quantitative diagnostic assays of MIC-1 in samples of maternal serum, amniotic fluid and placental extracts offers the possibility of detecting pregnant women with abnormal levels of MIC-1 and which are thereby at risk of miscarriage and/or premature birth.
In addition, the present applicants have found that a number of allelic variants of MIC-1 exist, all of which show minor amino acid sequence differences at positions 9, 48 and 202 (see International patent publication No. WO 97/00958, the entire contents of which is incorporated herein by reference, wherein MIC-1 is referred to as CL13). The most significant of these positions is amino acid position 202 since this corresponds to position 6 of the mature form of MIC-1 (i.e. with the leader sequence having been removed through cleavage). In some of the identified variants, the normal histidine (H) residue at position 202 (or “H6”) is substituted with aspartic acid (D). This is due to a single nucleotide substitution within the MIC-1 gene such that a cytosine (C) at position 604 is substituted by a guanosine (G). The present applicants have now recognised that subjects which are either heterozygous or homozygous for the Asp202-MIC-1 (or “D6”) allelic variant may have an altered predisposition and disease course for inflammatory disease(s) and/or cancer(s).